Page 74 - Haematologica - Vol. 105 n. 6 - June 2020

M.M. Majumder et al.
decline in drug metabolism and excretion,21,22 which can result in drug accumulation leading to unintended effects on other immune cells. Venetoclax displayed similar cell- specific effects in all tested samples, whether healthy or malignant, suggesting the variation in response is purely lineage specific.
Lineage specific effect of midostaurin on CD19+ cells is comparable to FLT3-mutated acute myeloid leukemia cells
In our primary screen, we observed selective depletion of B and NK cells in PB samples (n=3) treated with midostaurin (Figure 2A and Online Supplementary Figure S8A), which is approved for treating FLT3-mutated AML and systemic mastocytosis.23,24 To evaluate CD19+ cell specificity in malignant cells such as in CLL and to com- pare the response to FLT3-ITD-mutated AML cells, we tested midostaurin in 17 additional samples (Cohort II) derived from healthy (n=2), CLL (n=7), and AML patients with wild-type (WT) FLT3 (n=3) or harboring the FLT3- ITD mutation (n=5). Variable sensitivity was noted in the CD34+CD38- population, presumably leukemic stem cells, from all tested AML samples regardless of FLT3 mutation status (Figure 4A). CD34+CD38+/blast cells from all FLT3- ITD mutated AML samples were sensitive (median IC50, 554 nM) (Figure 4B). Remarkable sensitivity (IC50, 16 nM) was detected in the CD34+CD38+ fraction from one of the
three WT samples (Online Supplementary Figure S8B). While CD34+CD38- cells from healthy donors were insen- sitive, CD34+CD38+ cells from one healthy individual responded similarly to FLT3-ITD-mutated AML samples (Online Supplementary Figure S8B). Importantly, we observed high efficacy against CD19+ cells in all tested samples including those derived from CLL patients (Figure 4C, D and Online Supplementary Figure S8C) indicating a lineage specific effect. The effect on CD19+/B cells (medi- an IC50, 314 nM) was comparable to FLT3-ITD mutated AML CD34+CD38+ (blast) cells (Figure 4B and C). Our results suggest a need for further investigation to evaluate midostaurin efficacy in diseases affecting B-cell lineages such as CLL.
Characterizing protein abundance and basal cell signaling contributing to innate cellular response to therapies
Having determined lineage specificity of the tested small molecules, we next explored whether protein abun- dance or basal signaling profiles of specific cell popula- tions could explain the innate cellular responses. We also investigated whether healthy cells share identical basal activity for signaling proteins as patient-derived cells or whether basal intracellular signaling was deregulated dur- ing malignant transformation. To characterize and com- pare the proteomic background of healthy hematopoietic
ABC
D
Figure 4. Effect of midostaurin on the viability of CD34+CD38-, CD34+CD38+ and CD19+ cells derived from healthy donors, and acute myeloid leukemia (AML) or chronic lymphocytic leukemia (CLL) patients. Averaged dose response curves for disease categories are presented as mean±standard error of mean. (A) While midostaurin treatment had no effect on CD34+CD38- cells from healthy individuals, variable sensitivity was detected in AML samples. (B) CD34+CD38+ cells derived from FLT3-ITD-mutated AML samples displayed similar sensitivity (median IC50, 554nM). (C) CD19+ cells derived from healthy donor or patient samples showed com- parable sensitivity at a median IC50 of 319 nM. Individual dose response curves are provided in Online Supplementary Figure S8C. (D) Scatter plot showing dose responses for midostaurin in CD19+ cells from a CLL patient. The percentage of CD19+ live cells present in midostaurin-treated wells compared to untreated cells is displayed numerically on the plot. Cellular proportions for these samples are provided in Online Supplementary Table S5. Related IC50 values are provided in Online Supplementary Table S6.
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